Use of products of pcr amplification carrying elements of secondary structure to improve pcr-based nucleic acid detection

Abstract

Particular aspects comprise amplifying target nucleic acid using PCR and an oligonucleotide primer pair wherein at least one of the primers is designed to incorporate a 5'-specialty sequence to provide for an amplification product that intramolecularly folds into a secondary structure; and detecting the amplification product by a method comprising: providing an oligonucleotide cleavage component, hybridizing the oligonucleotide cleavage component with the amplification product to form a three-strand cleavage structure wherein two strands of the three-strand cleavage structure are provided by the secondary structure of the amplification product, cleaving 3′- or 5′-strands of the three-strand cleavage structure using a duplex-specific nuclease activity resulting in a cleavage product, and detecting the cleavage product indicative of the presence of the target nucleic acid. In certain aspects both primers incorporate a 5′-specialty sequence and detecting comprises cleaving 3′- or 5′-strands of a three-strand cleavage structure using duplex-specific nuclease to provide a cleavage product.